Application of Isoangustone A in preparation of antitumor drugs
Through the isolation and identification of the chemical components of licorice, Isoangustone A was found to have significant anti-tumor activity and was used to prepare anti-lung cancer and anti-hepatitis cancer drugs, which solved the problem of existing drug resistance and achieved effective inhibition and apoptosis induction of lung cancer and liver cancer cells.
Patent Information
- Application Number
- CN202510193547.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-21
- Publication Date
- 2025-05-06
AI Technical Summary
Existing anti-lung cancer and anti-liver cancer drugs are prone to drug resistance after long-term use, which is difficult to effectively inhibit the growth and metastasis of lung cancer and liver cancer cells.
Through systematic isolation, purification and structural identification of licorice chemical components, Isoangustone A was discovered and used as the active ingredient of anti-tumor drugs to prepare different dosage forms of drugs alone or in combination with other drugs, which were used to inhibit the proliferation of lung and liver cancer cells and induce apoptosis.
Isoangustone A significantly inhibits the proliferation of lung cancer cells A549 and liver cancer cells HepG2 and induces apoptosis, providing a scientific basis for the development of anti-tumor drugs, and has important application value.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of medical technology, and particularly relates to the use of Isoangustone A in preparing anti-tumor drugs. Background Art
[0002] Cancer is a serious public health problem in the world and a risk factor that seriously affects the health of residents. In the past decade, the global cancer burden has continued to grow. The main cancers that threaten the health of Chinese residents include lung cancer, liver cancer, breast cancer and stomach cancer. The incidence and mortality of lung cancer in my country are ranked first in the ranking of malignant tumors, of which non-small cell lung cancer accounts for about 80%. Surgical resection, chemotherapy and radiotherapy are the main treatments for lung cancer. Currently, targeted inhibitors such as crizotinib, gefitinib and erlotinib, which target epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK), have achieved significant therapeutic effects in clinical practice; however, after long-term use, these drugs have also developed drug resistance.
[0003] Compared with traditional chemical drugs, Chinese medicine has the advantages of multiple targets and less toxic side effects, which has attracted widespread attention. In recent years, a large number of clinical studies have confirmed that Chinese medicine has a certain therapeutic effect in anti-lung cancer. It can not only improve clinical symptoms, but also improve the quality of life and prolong the patient's survival with tumors. A variety of Chinese medicine extracts or their monomer components have a good inhibitory effect on the growth and metastasis of lung cancer cells. Licorice is a perennial herbaceous plant of the genus Glycyrrhiza in the Leguminosae family. It is a Chinese medicine that can be used as both medicine and food. The dried roots and rhizomes are used as medicine. It has the functions of strengthening the spleen and replenishing qi, clearing away heat and detoxifying, removing phlegm and relieving cough, relieving acute pain, and harmonizing various medicines. Literature reports that licorice is rich in flavonoids, coumarins, terpenes and their glycoside compounds, etc., and it has strong pharmacological activities in liver protection, anti-inflammatory, antioxidant and anti-cancer. Licorice is one of the main flavonoid components in licorice, and there is no report on its activity research. Summary of the invention
[0004] The purpose of the present invention is to provide a use of Isoangustone A in preparing anti-tumor drugs.
[0005] The present invention provides the use of the compound represented by formula A in the preparation of a drug for preventing and / or treating tumors: It is any one of Isoangustone A or its pharmaceutically acceptable ester, ether, salt, solvate, stereoisomer, tautomer and prodrug.
[0006] The tumor is lung cancer.
[0007] The tumor is liver cancer.
[0008] In the preparation of anti-tumor drugs, Isoangustone A is used through conventional preparation processes alone or in combination with other drugs to prepare various dosage forms of drugs that can be used clinically, including injections, tablets, capsules, soft capsules, microcapsules, nano preparations, granules, films, suppositories, aerosols and other dosage forms.
[0009] Compared with the prior art, the present invention has obvious beneficial effects. From the above technical scheme, it can be seen that the present invention systematically separates, purifies and structurally identifies the chemical components of licorice, and performs activity screening to find multiple compounds with significant biological activity. Isoangustone A has the activity of significantly inhibiting the proliferation of lung cancer cells A549 and liver cancer HepG2, and has the effect of inducing apoptosis of cancer cells, suggesting that it can be used for the treatment of tumors. The present invention provides a scientific basis for the development of anti-tumor drugs based on the Isoangustone A component, and has great application value. BRIEF DESCRIPTION OF THE DRAWINGS
[0010] Figure 1 is the chemical structural formula of Isoangustone A.
[0011] Figure 2 The crystal violet staining method was used to determine the inhibition rate of different concentrations of Isoangustone A on the proliferation of A549 and HepG2 cells.
[0012] Figure 3 Flow cytometry was used to detect the apoptosis of A549 cells induced by Isoangustone A.
[0013] Figure 4 Western blot was used to detect the effect of Isoangustone A on the protein expression of cleaved-PARP, Caspase3, Caspase-8, cleaved-Caspase3, Bax and Bcl-2 in A549 cells. DETAILED DESCRIPTION
[0014] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the use of the compounds of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified; the experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.
[0015] A method for preparing Isoangustone A comprises the following steps: Test Example 1: Preparation of Isoangustone A 20 kg of ulardia herb was crushed into coarse powder, extracted with 75% ethanol under heating and reflux for 3 times, each time for 2 hours, filtered, the extracts were combined, and concentrated under reduced pressure to obtain 2.8 kg of extract. The obtained extract was suspended in an appropriate amount of water, extracted with ethyl acetate, and the solvent was recovered under reduced pressure to obtain 460 g of ethyl acetate extract. Then, polyamide column chromatography was used, and gradient elution was performed using 10%, 30%, 50% and 90% ethanol as eluents, respectively. The eluents were concentrated and freeze-dried, and the concentrated samples were F1, F2, F3 and F4, respectively. The sample of part F3 was decolorized by small-pore resin column chromatography, then dissolved in an organic solvent, mixed with 80-mesh silica gel, separated by normal pressure silica gel column chromatography, and gradient eluted using dichloromethane-methanol, etc. After thin-layer chromatography detection, the eluents were combined to obtain 5 parts. Among them, the third part was repeatedly separated by forward and reverse silica gel column chromatography, and separated by semi-preparative high-performance liquid chromatography to obtain Isoangustone A (structural formula see Figure 1 ).
[0016] Isoangustone A spectrum data can be found in the reference: Ji S, Li Z, Song W, Wang Y, Liang W, Li K, Tang S, Wang Q, Qiao X, Zhou D, Yu S, Ye M. Bioactive Constituents of Glycyrrhizauralensis (Licorice): Discovery of the Effective Components of a Traditional Herbal Medicine. J Nat Prod. 2016 Feb 26; 79(2): 281-92.
[0017] Experimental Example 2: Inhibitory effect of Isoangustone A on A549 cells and HepG2 cells A549 cells were cultured in Ham's F12-K medium containing 10% FBS, and HepG2 cells were cultured in DMEM medium containing 10% FBS at 37°C and 5% carbon dioxide for adherent growth. The cells were inoculated in a 6-well culture plate. After 12 to 24 hours, the drug treatment could be performed when the cell confluence reached about 50%. Isoangustone A was diluted in fresh culture medium, and the original culture medium was replaced with fresh culture medium and cultured until the experimental design time. The culture medium was removed, and 2 mL of 1% glutaraldehyde solution was added to each well and fixed at room temperature for 15 minutes. After removing glutaraldehyde, PBS was rinsed twice, and 2 mL of 0.02% crystal violet solution was added to each well for staining for 30 minutes. The residual crystal violet was rinsed with distilled water, and 8 mL of 75% ethanol was added to each well. The well was placed on a decolorizing shaker and shaken until the crystal violet in the cells was completely dissolved. Finally, the absorbance value was measured at a wavelength of 590 nm using an enzyme reader. The wells without cells were used as blank controls, and the cell survival rate of the control group was 100%. The calculation formula is as follows:
[0018] A549 cells and HepG2 cells are relatively sensitive to Isoangustone A. The inhibitory effect of Isoangustone A on the growth of cancer cells is concentration-dependent. As the concentration of Isoangustone A increases, the survival rate of cancer cells decreases significantly (see Figure 2 shown).
[0019] Experimental Example 3: Isoangustone A induces apoptosis of A549 cells A549 cells were cultured in RPMI1640 medium containing 10% FBS at 37°C and 5% carbon dioxide, and adherently grown. The cells were inoculated in a 6-well culture plate, and after 12 to 24 hours, the drug treatment was performed when the cell confluence reached about 50%. Isoangustone A was diluted in fresh culture medium, and the original culture medium was replaced with fresh culture medium and cultured until the experimental design time.
[0020] Digest the adherent cells and collect them together with the suspended cells in a centrifuge tube, centrifuge at 1000g for 5 minutes at 4°C, remove the culture medium, rinse the cells with PBS and centrifuge again under the same conditions, remove the PBS to obtain the cells. Add 85μL of fixative, 10μL of PI and 5μL of AnnexinV-FITC to each sample, incubate at room temperature for 15 minutes, then add 400μL of fixative, and detect using the FL1 and FL2 channels of the flow cytometer within 1 hour. In the histogram, Q3 is Annexin V-PI double-negative cells, representing normal cells; Q4 is Annexin V single-positive cells, representing early apoptotic cells; Q2 is Annexin V-PI double-positive cells, representing late apoptotic cells; Q1 is PI single-positive cells, representing necrotic cells (see Figure 3 ).
[0021] Experimental Example 4: Isoangustone A acts on the intrinsic apoptosis pathway by upregulating cleaved-PARP, cleaved-Caspase 3, and Caspase-8 proteins, and downregulating the Bcl-2 / Bax protein ratio. A549 cells were cultured in Ham's F-12K medium containing 10% FBS at 37°C and 5% carbon dioxide, and adherent growth was achieved. The cells were inoculated in a 6-well culture plate, and drug treatment was performed after 12 to 24 hours when the cell confluence reached about 50%. Isoangustone A was diluted in fresh culture medium, and the original culture medium was replaced with fresh culture medium and cultured until the experimental design time.
[0022] Collect adherent and suspended cells, add appropriate amount of RIPA lysis buffer (containing protease inhibitors), lyse on ice for 15-30 minutes, then ultrasonically disrupt, centrifuge at 15000rpm, 4℃ for 30 minutes. Take the supernatant of the centrifuged sample to measure the total protein concentration by BCA method. Use lysis buffer to adjust the total protein concentration of each sample to the same, add loading buffer in proportion, mix well, and heat in a boiling water bath for 5-10 minutes for denaturation treatment.
[0023] Select the concentration of separation gel according to the molecular weight of the protein to be detected, prepare separation gel and integration gel, add protein sample and protein marker for electrophoresis. The sample is electrophoresed at a voltage of 80V in the integration gel. After the sample enters the separation gel, the voltage is adjusted to 100V until the front end of the bromophenol blue indicator reaches the bottom of the separation gel, and the electrophoresis is terminated. The protein sample on the separation gel is electrotransferred to the PVDF membrane by wet transfer, and the membrane is transferred at 100V for 120min or at 30V overnight. The membrane should be kept at a low temperature.
[0024] After the transfer is completed, the PVDF membrane is blocked with PBS containing 5% to 10% skim milk powder at room temperature for 1 hour. After blocking, incubate with the antibody of the target protein for 1 to 2 hours (or block overnight at 4°C). After the primary antibody incubation is completed, rinse the PVDF membrane with PBS 3 times, 5 minutes each time, then incubate with the secondary antibody for 1 to 2 hours, and rinse with PBS 3 times, 10 minutes each time. In the dark room, ECL luminescence color development, X-ray film exposure, development, and fixation can appear on the X-ray film. The abundance of the band is the level of expression of the corresponding protein in the sample.
[0025] The expression levels of apoptosis pathway-related molecules were detected by Western blot (see Figure 4 ). Compared with the control group (Control), after A549 cells were treated with 10μM Isoangustone A for 72h, Isoangustone A could significantly upregulate the expression level of cleaved-PARP protein (P<0.05); when the concentration of Isoangustone A reached 5μM, the ratio of Bcl-2 to Bax was significantly reduced (P<0.05); and the expression levels of caspase 8 (P<0.05) and cleaved-Caspase3 (P<0.001) were significantly upregulated.
[0026] In summary, Isoangustone A has significant anti-tumor effect and has good development and application prospects.
[0027] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still falls within the scope of the technical solution of the present invention.
Claims
1. Use of Isoangustone A in the preparation of anti-tumor drugs, The compound structure of licorice root A (CAS No.: 129280-34-8) is shown in formula (I): Isoangustone A or any one of its pharmaceutically acceptable salts, stereoisomers and tautomers; The tumor is lung cancer; The tumor is liver cancer.
2. The use of Isoangustone A in a drug for treating lung cancer according to claim 1, wherein the lung cancer cells are A549 cells.
3. The use of Isoangustone A in a drug for treating liver cancer according to claim 1, wherein the liver cancer cells are HepG2 cells.
4. The method for preparing Isoangustone A according to claim 1, comprising the steps of: 1) 20 kg of ulardia herb was crushed into coarse powder, extracted with 75% ethanol under heating and reflux for 3 times, each time for 2 h, filtered, the extracts were combined, and concentrated under reduced pressure to obtain 5.8 kg of extract; 2) The extract obtained above was suspended in an appropriate amount of water, extracted with ethyl acetate, and the solvent was recovered under reduced pressure to obtain 650g of ethyl acetate extract. Then, polyamide column chromatography was used to perform gradient elution using 10%, 30%, 50% and 90% ethanol as eluents, and the eluent was concentrated. The concentrated samples were divided into four parts: F1, F2, F3 and F4; 3) The sample of part F3 was decolorized by small-pore resin column chromatography, dissolved in an organic solvent, mixed with 80-mesh silica gel, separated by normal pressure silica gel column chromatography, gradient eluted with dichloromethane-methanol, etc., and the eluents were combined after thin-layer chromatography detection to obtain 5 parts. Part 3 was separated repeatedly by forward and reverse silica gel column chromatography, and separated by semi-preparative high performance liquid chromatography to obtain Isoangustone A.